Human group A rotavirus, strain 69M, isolated from an ill child in Indonesia, has been described as a new serotype (serotype 8). Previous sequence analyses in our laboratory of the gene encoding the outer capsid protein VP7 confirmed that 69M is distinct from established human rotavirus serotypes 1, 2, 3 and 4. In order to assess whether, VP4 (encoded by gene 4), the other major outer capsid neutralization protein of 69M is also distinct, the complete nucleotide sequence of the gene 4 was determined. The sequence of the 69M VP4 gene was compared with available VP4 gene sequences from human and animal rotaviruses. In addition, Northern blot hybridization was performed to examine the genetic relatedness of the 69M VP4 gene with other group A rotaviruses using 32p-labeled cloned 69M VP4 gene DNA or 69M ssRNA transcript probes. These genetic studies as well as neutralization confirmed that the fourth gene (encoding VP4) of strain 69M is different from any of the other corresponding genes described thus far. Of interest, comparative analyses of the deduced amino acid sequences suggest that the VP4 of 69M is more closely related to animal rotaviruses than to human rotaviruses.